Changes in macromolecule syntheses under special consideration of UNA and the energy providing system in imbibing embryos of different agedagrostemma githago L. Seeds

Changes in macromolecule syntheses, especially RNA synthesis, and the energy providing system were investigated in seeds ofAgrostemma githago aged for different periods. In embryos of aged seeds all macromolecule syntheses start later and reach a lower level than young ones. It was found that the synthesis of rRNA in embryos of aged seeds is reduced whereas the synthesis of poly (A⁺) RNA in relation to the total RNA synthesis is highly increased as well as the amount of this RNA species with long poly (A) chains. The results are discussed in connection with the decreased protein synthesis and the reduced ATP content and ATP formation ability in embryos during the long time storage of seeds.     

PVA和PEG(6000)预处理对大豆胚轴生长、蛋白质合成和ATP含量的影响

在低温吸胀阶段,经PVA(聚乙烯醇)和PEG(聚乙二醇6000)预处理的大豆胚轴蛋白质合成和ATP含量均比对照高。在萌发阶段,胚轴生长增快,蛋白质合成明显加快,ATP迅速被消耗,而对照胚轴则相反。试验结果表明,预处理大豆种子萌发和生长与其蛋白质合成、ATP水平和消耗能力有密切关系。     

Untersuchungen über RNA- und proteinbiosynthesen in aktivierten und temperaturblockiertenAgrostemma githago-samen: einfluss von benzylaminopurin

Embryos ofAgrostemma githago seeds blocked by higher temperature are suitable objects for the study of the regulation of gen expression independent of transeriptional processes. Germination of afterripened seeds can be prevented by imbibing at 30°C, whereas at 20°C germination will be completed after 30 h of soaking (BORRISS 1956). In temperature-blocked embryos RNA is synthesized with undiminished intensity, whereas DNA and protein syntheses are remarkably reduced. Equal amounts of poly(A)-RNA (probably mRNA), enriched by affinity chromatography on oligo(dT)-ceIlulose columns, are detected in blocked and activated embryos. We suggest that in blocked embryos not synthesis itself, but the availability of mRNA to polysomal fraction is reduced. Benzylaminopurine is able to break secondary dormancy ofAgrostemma githago seeds. In benzylaminopurine-treated seeds there is only a slight increase of the incorporation of¹⁴C-L-leu-cine in protein.     

菜用大豆种子活力与DNA,RNA及蛋白质合成的关系

菜用大豆种子随着其活力的下降,对DNA,RNA和蛋白质前体的吸收,以及合成这些大分子的能力都明显下降,已丧失合成DNA和蛋白质能力的失活种子,仍能进行微弱的RNA合成。高活力种子在吸胀初期DNA合成速率较低,然后增加,至16h达高峰;RNA的合成速率在吸胀一开始就很高,在整个吸胀过程中均保持较高水平;蛋白质的合成速率则在开始较高,并随着吸胀过程呈增强趋势。     

Inhibition of pregerminative processes in dormant and thermodormant Agrostemma githago seeds

Imbibed non-dormant seeds do not germinate immediately after completion of water uptake and reactivation of their metabolism. During the lag-period the seeds apparently undergo processes which are essential for germination. The extent to which these pregerminative processes occur in dormant seeds (freshly harvested seeds) and thermodormant seeds (afterripened seeds imbibed at a supra-optimal temperature) of Agrostemma githago was determined. The pregerminative processes were inhibited almost completely in dormant seeds, but only to 50% or less in thermodormant seeds. When seeds were progressing through the pregerminative processes, the axes showed a higher rate of protein synthesis than axes of blocked seeds. However, this increased rate of protein synthesis was a late event and neither necessary nor sufficient for germination.     

On the Role of Stored mRNA in Protein Synthesis in Embryonic Axes of Germinating Vigna unguiculata Seeds

Polyadenylated (poly A⁺) RNAs were prepared from both dry and incubated embryonic axes of Vigna unguiculata seeds and were translated by a wheat germ translation system. Analysis with gel electrophoresis and fluorography showed that translation products of poly A⁺ RNA from dry embryonic axes were nearly the same as those from 2-hour incubated axes but somewhat different from those of 4- to 24-hour incubated axes, and that translation products remained almost unchanged between the 4- and 24-hour stages of postimbibition. The results indicate the possibility that the stored mRNA (poly A⁺ RNA from dry embryonic axes) directs the protein synthesis required for early stages of germination. This is supported by comparison of the in vitro translation products of poly A⁺ RNAs with those of polysomal RNAs. Experiments with α-amanitin, a specific inhibitor of RNA polymerase II (J. Jendrisak 1980 J Biol Chem 255: 8529-8533), suggested that the synthesis of some of the stored mRNA species is resumed as early as 4 hours after the onset of imbibition.     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

The influence of poly(A)-binding proteins on translation of poly(A)+ RNA in a cell-free system from embryo axes of dry pea seeds

Ribonucleoproteins of the ribosomal fraction of germinated pea embryo axes, containing translationally active mRNA, differ from analogous ribonucleoproteins of dry pea seeds, which contain stored mRNA, by the presence of a 60 kDa protein fraction showing affinity to poly(A). The above protein fraction largely affects the activity of poly(A)⁺ RNA translation in cell-free system. An activating effect is clearly seen at a weight ratio of poly(A)-binding proteins:poly(A)⁺ RNA of 3:1, whereas with an increase in the concentration of these proteins the translational activity drops. The effect of poly(A)-binding proteins containing the 60 kDa fraction on poly(A)⁺ RNA dependent cell-free translation can be efficiently reduced by simultaneous addition of synthetic poly(adenylic acid). It was also proved that activation of translation does not influence its products. It is concluded that poly(A)-binding proteins from the ribosomal fraction of embryo axes of pea seeds, especially the 60 kDa fraction, are involved in regulation of the translational activity of poly(A)⁺ RNA.     

Wogonin induces differentiation and neurite outgrowth of neural precursor cells

Currently, [³H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [α-³³P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1α or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2α. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2α knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.     

Polyadenylation of pea seed RNA at the early stages of germination.

1. The RNA polyadenylating activity was found in embryo axes of dry, as well as imbibed and germinated pea seeds, both in nucleus and cytoplasm. 2. The total enzymatic activity remains unchanged during germination, but the intracellular distribution is altered; the activity in nuclei is increased about four-fold at the expense of the postmitochondrial fraction. 3. Specificity towards RNA primers of the polyadenylating system from pea embryo axes is low. 4. Cordycepin inhibits RNA polyadenylation only when [14C]ATP is used as a nucleotide donor, and has no visible influence on the activity of the system utilizing [14C]oligo(A)-nucleotides. 5. It seems that RNA in the pea embryo axes is polyadenylated by a two-step mechanism: synthesis of oligo(A)-nucleotides, and their addition to RNA.     

Desiccation of Axes of Phaseolus vulgaris during Development of a Switch from a Development Pattern of Protein Synthesis to a Germination Pattern

Immature seeds of Phaseolus vulgaris removed from the pod at 32 days of development do not germinate unless first subjected to a desiccation treatment. This change from development to germination caused by premature drying is mirrored in the pattern of protein synthesis by the axes. Rehydrated axes from 32-day-developed seeds cease to synthesize proteins that are uniquely associated with development, but instead synthesize some proteins that are similar to those made in the germinating axes from mature dry seeds. Desiccation of 22-day-developed seeds does not lead to their germination, nor does it cause a switch from a developmental to a germination mode of protein synthesis by the axes. It is proposed that desiccation plays a role in permanently suppressing developmental protein synthesis and in inducing germination protein synthesis.     

Protein degradation during sporulation ofBacillus megaterium: Effect of actinomycin D

The first acceleration of protein degradation in cells ofBacillus megaterium was found at the stage 0–I of sporulation, the second one at the stage II–III, where the sporulation process became irreversible. These accelerations were reduced by actinomycin D inhibiting RNA and protein syntheses by more than 95%. In the presence of the antibiotic, only 8% of prelabeled proteins were degraded. Actinomycin D did not lower either the concentration of ATP or the proteolytic activity in the homogenate prepared from sporulating cells. This indicates that the inhibition of protein catabolism by actinomycin D was not owing to the absence of ATP or proteolytic enzymes. Actinomycin probably inhibited an unknown step preceding the proteolytic attack of the protein molecules during sporulation, because it had no significant effect on proteolysis during vegetative growth.     

Differential responses of Mimusops elengi and Manilkara zapota seeds and embryos to cryopreservation

Several dehydration protocols were evaluated for their ability to cryopreserve intact seeds and excised embryonic axes of Mimusops elengi and Manilkara zapota (Sapotaceae). Both interspecific and intraspecific variations in cryotolerance were found. M. zapota embryonic axes were more tolerant of cryopreservation than those of M. elengi, and showed higher desiccation tolerance, higher post-thawing survival and development, and a much wider range of moisture contents for cryopreservation. Maximum development rates were 94% and 27% for M. zapota and M. elengi, respectively. Intact seeds of both species tolerated desiccation to low moisture levels, but were sensitive to liquid nitrogen exposure, and cryopreserved seeds failed to germinate. Assessment of developing embryos excised from cryopreserved seeds associated nonviability of cotyledons and plumules with germination failure. Other structures survived at variable rates; most hypocotyls and radicles (up to 76% and 98% for M. elengi and M. zapota, respectively) were viable. The different cryotolerance between hypocotyls and cotyledons is a critical cause for failure in cryopreservation, contributing to the difficulty in developing protocols for such intermediate oily seeds.     

Abscisic Acid Levels in Soybean Reproductive Structures during Development

Abscisic acid (ABA) concentrations and growth rates of developing soybean (Glycine max [L.] Merr. cv. Wye) seeds and pod walls were determined from anthesis to maturation using high pressure liquid chromatographic techniques. Developing soybean seeds contain up to 12,200 ng/g fresh weight of ABA compared to 330 ng/g fresh weight for pod walls. In the developing seeds ABA levels correlated with growth rates, being the highest during the most active growth period of seed enlargement, and then decreasing to less than 10 ng/g fresh weight at maturity. Higher levels of ABA were found to occur in the cotyledons and seed coats than the root-shoot axes at 21 days postanthesis. The time required for excised root-shoot axes to initiate growth in liquid culture decreased as seed development progressed and ABA levels of the seeds declined.     

Potassium-Dependent Increase in RNA and Protein Synthesis in the Early Phase of Incubation of the Thermodormant Phacelia tanacetifolia Seeds

The seeds of Phacelia tanacetifolia Benth. cv Bleu Clair incubated at 30°C in the dark did not germinate and did not activate K⁺ uptake capacity. The administration of 1 millimolar K⁺ in the early phase of incubation stimulated RNA and protein synthesis. The possible role of K⁺ in promoting the marcromolecular syntheses during the early phase of germination is discussed.     

Sugars and desiccation tolerance in seeds

Soluble sugars have been shown to protect liposomes and lobster microsomes from desiccation damage, and a protective role has been proposed for them in several anhydrous systems. We have studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating soybean (Glycine max L. Merr. cv Williams), pea (Pisum sativum L. cv Alaska), and corn (Zea mays L. cv Merit) axes. The loss of desiccation tolerance during imbibition was monitored by following the ability of seeds to germinate after desiccation following various periods of preimbibition and by following the rates of electrolyte leakage from dried, then rehydrated axes. Finally, we analyzed the soluble sugar contents of the axes throughout the transition from desiccation tolerance to intolerance. These analyses show that sucrose and larger oligosaccharides were consistently present during the tolerant stage, and that desiccation tolerance disappeared as the oligosaccharides were lost. The results support the idea that sucrose may serve as the principal agent of desiccation tolerance in these seeds, with the larger oligosaccharides serving to keep the sucrose from crystallizing.     

Ethanol and acetaldehyde in imbibing soybean seeds in relation to deterioration

Deterioration as evidenced by decline in germination or seedling growth of soybean (cv. Essex) seeds during accelerated aging treatments at 41 C and 100% relative humidity is accompanied by increased levels of acetaldehyde and ethanol in imbibing embryonic axes and seeds. These increases become more pronounced with duration of the aging treatment. A similar inverse relationship between levels of acetaldehyde and ethanol and deterioration was observed when seeds were “naturally” aged for several years. During imbibition of low-vigor, accelerated-aged seeds at 25 C, acetaldehyde and ethanol increased from near trace amounts in dry tissues to maximum levels at 4 hours. Increases in acetaldehyde and ethanol during imbibition were less in high- than in low-vigor seeds. Increases were also less pronounced in low-vigor seeds when water uptake injury was avoided by osmotically decreasing water uptake rate with 30% polyethylene glycol. Embryonic axes from deteriorated seeds were characterized by low rates of O₂ uptake and high respiratory quotients relative to the unaged controls. Anaerobic conditions and respiratory inhibitors, such as sodium azide, increased acetaldehyde and ethanol in unaged seeds to levels similar to those in accelerated-aged seeds after 2 hours imbibition. It is suggested that, during aging, an imbalance between tricarboxylic and glycolytic activities, present during early imbibition to some degree even in vigorous unaged seeds, becomes more pronounced and leads to accumulation of ethanol and acetaldehyde.     

Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds

Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech (Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 °C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage.